Composition for treating cancer associated with hpv infection

ABSTRACT

The present invention relates to a composition for preventing or treating diseases associated with human papillomavirus (HPV), and more specifically, cancer associated with HPV, and even more specifically, cervical cancer. The nucleotide sequence of the present invention, the sequence in which the base thereof is modified, and a specific combination thereof can be useful in a composition for effectively treating diseases associated with HPV infection by greatly inhibiting the expression of the E6/E7 gene of HPV type 16 or 18.

TECHNICAL FIELD

This patent application claims priority of Korean Patent Application No.10-2012-0084820, filed on Aug. 2, 2012, the entire content of which ishereby incorporated by reference.

The present invention relates to a composition for gene therapy for adisease associated with HPV infection including cervical cancer.

BACKGROUND ART

High-risk human papilloma virus (hereinafter, “HPV”) types and 18 aremajor factors of cervical cancer and cervical dysplasia, and becomecauses of other genital cancers and a head and neck squamous cancer.Cervical cancer is one of the most general types of malignant tumors ofwomen. Although the incidence of invasive cervical cancer has beenslowly reduced, the invasive cervical cancer is a still most frequentcancer of women in developing countries, which holds 25% of femalecancers. HPV is a small DNA virus having approximately 8,000 basesequences which causes benign or malignant tumors. So far, depending ona genomic difference, at least about 100 HPV subtypes have beenidentified, and genotypes of approximately HPVs have been completelyanalyzed. Among these types, high-risk HPV types (e.g., HPV-16, 18, 31,33, 35, 45, 51, 52, and 56) relate to almost 90% of cervical cancer. Atleast 50% of cervical cancer infected with HPV relate to HPV type 16,and followed by HPV type 18 (12%), HPV type 45 (8%), and HPV type (5%).These HPVs encode 2 oncogenic proteins, which are, protein E6 and E7.Both proteins are involved in HPV-mediated cell immortalization and celltransformation. The oncogenic E6 protein binds to wild-type p53 tumorsuppressor protein to thereby degrade p53 through an ubiquitin pathway.On the other hand, the E7 protein directly binds to Rb to therebyoverphosphorylate Rb. At first, E6 forms a complex with an E6-associatedprotein (E6-AP) which is an E3 ubiquitin-protein ligase. Then, theE6/E6-AP complex binds to and ubiqutinate wild-type p53, and theninterferes with p53-mediated cellular reaction to DNA damage. Mostly,the p53 tumor suppressor protein is regulated by Mdm2-mediatedubiquitination, however, in HPV-infected cervical cancer cells,degradation of p53 is completely changed to E6-mediated ubiquitinationfrom Mdm2-mediated ubiquitination. Thus, unlike many other cancers, mostcases of HPV-infected cervical cancer have the wild-type p53 gene.However, an expression level of the p53 protein is very low due to theconsistent degradation by the E6 protein. Particularly, the HPV E6protein has been significantly noticeable as a specific target forkilling just cervical cancer cells. These strategies, targeting to E6 orthe E6/E6-AP complex, include various treatment.

Examples include: use of a cellular toxin agent, an inhibitor to releasezinc of the E6 oncogenic protein, an epitope peptide (mimotope)mimicking E6-AP, anti-E6 ribozyme, a peptide aptamer which is targetedto the E6 oncogenic protein of a virus, siRNA which is targeted to theE6 oncogenic protein of the virus, and a combined treatment thereof.Recently, it has been proven that siRNA selectively silences anintrinsic gene in animal cells, and as well as, selectively silences aviral gene in a disease caused by a virus. RNA interference (RNAi) dueto transfection of siRNA has been emerged as a novel therapy fortreating viral infection of the human. siRNA, which is targeted to E6and E7 genes in HPV-infected cervical cancer cells, causes p53 and pRbaccumulation which leads to apoptosis or cell senescence. For an HPV-16infected cervical cancer cell line and an HPV-18 infected cell line, ithas been found that RNAi, which is targeted to E6 and E7 oncogenes ofviruses, selectively sciences expression of these proteins.

Meanwhile, efficacy of a nucleic acid having various modifications fornucleic acids (for example, in a base, a sugar and/or phosphate) isenhanced by inhibiting degradation caused by serum ribonuclease. Severalexamples describing sugar, base and phosphate modifications, which maybe introduced to a nucleic acid, are known in the art. For example, anoligonucleotide is modified to enhance stability and/or enhance thebiological activity through a modification by a nuclease-resistantgroup, for example, through 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, and 2′-H nucleotide base modification (see Eckstein et al.,PCT Laid-open Publication WO 92/07065; document [Perrault et al., Nature344:565-568, 1990]; document [Pieken et al., Science 253: 314-317,1991]; document [Usman and Cedergren, Trends in Biochem. Sci. 17:334-339, 1992]; Usman et al. PCT Laid-open Publication WO 93/15187;Sproat, U.S. Pat. No. 5,334,711 and document [Beigelman et al., J. Biol.Chem., 270:25702, 1995]; Beigelman et al., PCT Laid-open Publication WO97/26270; Beigelman et al., U.S. Pat. No. 5,716,824; Usman et al., U.S.Pat. No. 5,627,053; Woolf et al., PCT Laid-open Publication WO 98/13526;Thompson et al., U.S. Patent Application No. 60/082,404 (filed on Apr.20, 1998); document [Karpeisky et al., Tetrahedron Lett., 39:1131,1998]; document [Earnshaw and Gait, Biopolymers (Nucleic acid Sciences)48:39-55, 1998]; document [Verma and Eckstein, Annu. Rev. Biochem.67:99-134, 1998]; and document [Burlina et al., Bioorg. Med. Chem. 5:1999-2010, 1997]). Similar modifications may be used to modify thenucleic acid of the present invention.

In 1999, as a result of combined therapy of cisplatin-based chemotherapyand radiotherapy, survival rate of women who have a severe cervicalcancer in local has been significantly improved. Currently, cisplatin isa DNA-damaging drug which is widely used to treat cancers includingovarian, cervical, head and neck, non-small cell lung cancers and soforth. More recently, a working mechanism of a medicine based onplatinum has been investigated. However, it is still not fullyunderstood about a process in cells including DNA repair, cell death,cell cycle trajectory, signaling of DNA damage, and regulation inabsorption and secretion of a drug due to cisplatin treatment. In HPV-18HeLa cells, the p53 protein is escaped from E6-mediated degradation andpreferentially accumulated in nucleolus of a nucleus after cisplatintreatment. Also, HPV-16 SiHa cells recover p53 function by simultaneousradiotherapy and cisplatin treatment, thereby increasingradiosusceptibility.

Therefore, as a result of attempting to investigate an effective siRNAhaving a novel sequence by imparting a chemical modification toE6/E7-specific siRNA so that the siRNA may show the anti-cancer effect,alone or in a complex combination, or show a synergistic effect whenperforming combination therapy with conventional chemotherapy orradiotherapy, the present inventors have found that nucleotides listedon following Examples and Claims and particular combinations thereofreduce expression of relating proteins TP53 and E7, and HPV E6 mRNA, andinduce cell death, and also experimentally proven that efficacy achievedwhen used alone or in combination with anti-cancer agents is much betterthan that of RNA, which does not have a base sequence residuemodification.

Throughout the specification, numerous journals and patent documents arereferenced, and the citation is indicated. Disclosures of cited journalsand patent documents are incorporated herein in their entireties byreference to more clearly describe the level of the technical field towhich the present invention belongs and features of the presentinvention.

DISCLOSURE OF THE INVENTION Technical Problem

The present inventors continuously study and try to develop an efficientgene therapeutic agent for various diseases caused by human papillomavirus (HPV) infection. As a result, the present invention has beencompleted by finding that when using particular RNA for inhibitingexpression, which is targeted to an E6/E7 gene of HPV type 16 or HPVtype 18 virus, or a RNA sequence having a modification in a base of theRNA, HPV gene expression is efficiently inhibited, to thereby show anexcellent therapeutic activity on diseases associated with HPV infectionincluding a cervical cancer.

One object of the present invention is to provide a composition forpreventing or treating a disease associated with HPV infection, moreparticularly, an HPV infection-associated cancer, and further moreparticularly a cervical cancer.

Another object of the present invention is to provide a method forpreventing or treating a disease associated with HPV infection, moreparticularly, an HPV infection-associated cancer, and further moreparticularly a cervical cancer.

Other objects and benefits of the present invention become clear byappended detailed description of the invention, claims, and drawings.

Technical Solution

According to one aspect of the present invention, the present inventionprovides a composition for treating or preventing a disease associatedwith human papilloma virus (HPV) infection, the composition including,as an active ingredient, one or more nucleotide sequences selected fromthe group consisting of sequences of SEQ ID NOs: 16, 22, 28, 34, 40, 66,72, 84, 90, and 108, and antisense nucleotide sequences thereof.

The present inventors continuously study and try to develop an efficientgene therapeutic agent for various diseases caused by HPV infection.Consequently, it has been found that when using particular RNA forinhibiting expression, which is targeted to E6/E7 genes of HPV type 16or HPV type 18 virus, or a RNA sequence having a modification in a baseof the RNA, HPV gene expression is efficiently inhibited to thereby showan excellent therapeutic activity on diseases associated with HPVinfection including cervical cancer.

According to the present invention, sequences of SEQ ID Nos: 16, 22, 28,34, and 40 and sequences of SEQ ID Nos: 72, 84 90, and 108 are RNAnucleic acid sequences for inhibiting expression, wherein sequences ofSEQ ID Nos: 16, 22, 28, 34, and 40 are targeted to HPV type 16 virus;and sequences of SEQ ID Nos: 72, 84 90, and 108 are targeted to HPV type16 virus.

As used herein, the term “nucleotide” means a ribonucleotide present ina single strand or a double strand form, and includes a naturalnucleotide analogue unless otherwise specifically indicated (see Scheit,Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman,Chemical Reviews, 90:543-584(1990)).

As used herein, the term “inhibition of expression” means to leaddecline in a function of a target gene, and preferably means thatexpression of the target gene become undetectable or resultantly existsat the meaningless level

According to a preferred embodiment of the present invention, thenucleotide sequence of the present invention is an RNA sequence havingan ability to silence E6/E7 genes of HPV type 18 or HPV type 16 virus,and preferably, siRNA, shRNA, or an antisense oligonucleotide.

As used herein, the term “siRNA” means short double chain RNA which mayinduce RNA interference (RNAi) through cleavage of particular mRNA. ThesiRNA consist of a sense RNA strand having a homologous sequence withmRNA of a target gene, and an antisense RNA strand having acomplementary sequence thereto. siRNA can inhibit expression of thetarget gene, and is thus provided as an efficient gene knock-down methodor a gene therapy method.

siRNA is not limited such that siRNA having a double chain RNA part withRNA pairs is completely paired; rather includes a part which does notform a pair because of mismatch (i.e., a corresponding base is notcomplementary), bulge (i.e., there is no base corresponding to one-sidechain), and so forth. The total length is 10 to 100 bases, preferably 15to 80 bases, and most preferably 17 to 23 bases. Both a blunt end and acohesive end are available as an siRNA end structure, if it is possibleto inhibit target gene expression by the RNAi effect. For the cohesiveend structure, both a 3 end protrusion structure and a 5 end protrusionstructure are available. The number of protruded bases is not limited.For example, the number of bases may become 1 to 8 bases, and preferably2 to 6 bases. In addition, siRNA may include, for example, low-molecularweight RNA (e.g., natural RNA molecules such as tRNA, rRNA, and viralRNA, or synthetic RNA molecules) in a protrusion part of one end in therange where the effect of inhibiting target gene expression may beretained. In the end structure of siRNA, it is not necessary to have acleavage structure in both sides, and the structure may be a stem-loopstructure in which an end-portion of one side of double chain RNA isconnected by linker RNA. A length of the linker is not particularlylimited unless the length affects paring in a stem part.

As used herein, the term “small hairpin RNA (shRNA)” means 50 to 70single-stranded nucleotides, and forms the stem-loop structure in vivo.In other word, shRNA is a RNA sequence to form a tight hairpin structureto inhibit gene expression through RNA interference. A double strandstem is formed by base-pairing of long RNA having 15 to 30 complementarynucleotides at both sides of a loop site having 5 to 10 nucleotides. Forconstitutive expression, shRNA is transduced into cells through a vectorincluding U6 promoter, and mostly transferred to daughter cells tohereditary transmit inhibition of gene expression. The shRNA hairpinstructure is cleaved by an intracellular mechanism to become siRNA, andthen binds to an RNA-induced silencing complex (RISC). These RISCs bindto and cleave mRNA. shRNA is transcribed by RNA polymerase □. Accordingto the present invention, the nucleotide sequence of the presentinvention may form the shRNA structure having a double strand stemsequence at both sides of the loop site.

As used herein, the term “microRNA (miRNA)” means a single strand RNAmolecule which regulates gene expression and includes 10 to 50nucleotides in full-length, preferably 15 to nucleotides, and morepreferably 17 to 25 nucleotides. miRNA is an oligonucleotide which isnot expressed in cells and has a short stem-loop structure. miRNA isfully or partially homologous with at least one messenger RNA (mRNA),and inhibits target gene expression by complementarily binding to themRNA.

As used herein, the term “antisense oligonucleotide” means RNAcontaining a nucleotide sequence complementary to a particular mRNAsequence, or a derivative thereof, and inhibits translation of mRNA intoa protein by binding to the complementary sequence in mRNA. Theantisense nucleotide sequence of the present invention means an RNAsequence that may be complementary to mRNA of a target gene to bind tomRNA of the target gene, and may inhibit translation of the target geneinto mRNA, translocation into cytoplasm, maturation, or other essentialactivities for overall biological functions.

To enhance efficacy of the antisense oligonucleotide, a modification maybe made at a position of one or more of bases, sugars or backbones (seeDe Mesmaeker et al., Curr Opin Struct Biol., 5(3):343-55, 1995). Theoligonucleotide backbone may be modified with phosphorothioate,phosphotriester, methyl phosphonate, single-chain alkyl, cycloalkyl,single-chain heteroatomic, heterocyclic sugar sulfonate, and so forth.In addition, an antisense nucleic acid may include one or moresubstituted sugar moieties. The antisense oligonucleotide may include amodified base. Examples of modified bases include hypoxanthine, 6-methyladenine, 5-methyl pyrimidine (particularly, 5-methyl cytosine),5-hydroxymethyl cytosine (HMC), glycosyl HMC, gentobiosyl HMC,2-aminoadenine, 2-tiouracil, 2-tiothymine, 5-bromouracil,5-hydroxymethyl uracil, 8-azaguanin, 7-deazaguanin,N6(6-aminohexyl)adenine, 2,6-diaminopurine, 2-O-methyl uracil,2-O-methylguanin, 2-fluorocytisine, and so forth.

According to a more preferred embodiment of the present invention, thenucleotide sequence of the present invention is an siRNA sequence.

According to another aspect of the present invention, the presentinvention provides a composition for treating or preventing a diseaseassociated with HPV infection, the composition including, as an activeingredient, one or more nucleotide sequences selected from the groupconsisting of sequences of SEQ ID NOs: 1, 7, 12, 16, 22, 28, 34, 40, 46,51, 56, 62, 66, 72, 78, 84, 90, 96, 102 and 108, and antisensenucleotide sequences thereof, which have a modified backbone or one ormore modified bases. In other word, a nucleotide having the nucleotidesequences listed above, in which a backbone or a base is modified, maybecome the composition of the present invention for preventing ortreating a disease associated with HPV infection.

Modifications of a backbone or a base to be applied to the nucleotide ofthe present invention may include any modification which isconventionally employed in the art to increase stability or the desiredactivity.

Preferably, the modified backbone of the present invention includes oneor more modifications selected from the group consisting ofalkylphosphonate, phosphorothioate, phosphorodithioate,alkylphosphonothioate, phosphoamidate, phosphate ester, carbamate,acetamidate, carboxymethyl ester, carbonate, and phosphate triester.

Preferably, the modified base of the present invention includes one ormore modifications selected from the group consisting of methylation,glycosylation and halogenation. More preferably, the modified base ofthe present invention is a 2′-O methylated or a 2′-fluorinated base.

According to the present invention, when imparting a modification suchas 2′-O methylation or 2′-fluorination to a particular position of RNAtargeted to E6/E7 genes of HPV type or HPV type 18 virus for inhibitingexpression, comparing with an unmodified nucleic acid molecule, thepresent inventors have found that: efficiency of inhibiting target geneexpression is remarkably increased; stability in human serum isincreased; and half-life is considerably increased by at least two timesin a pharmacokinetic experiment in animals.

2′-O methylation means that a hydroxyl group attached to carbon numbertwo of ribose of an RNA molecule is methylated, and thus modified to a2′-methoxy group; and 2′-fluorination means that the hydroxyl groupattached to carbon number two of ribose of the RNA molecule issubstituted with a fluoro group and thus modified to a 2′-fluoro group.

According to a preferred embodiment of the present invention, the 2′-omethylated base in the nucleotide of the present invention is U or G.

According to a preferred embodiment of the present invention, the2′-fluorinated base in the nucleotide of the present invention is C.

According to a preferred embodiment of the present invention, thenucleotide sequence of the present invention having one or more of the2′-O methylated or the 2′-fluorinated base is selected from the groupconsisting of sequences of SEQ ID NOs: 2, 3, 5, 6, 8, 10, 11, 13, 15,17, 18, 20, 21, 23, 24, 26, 27, 29, 30, 32, 33, 35, 36, 38, 39, 41, 42,44, 45, 47, 49, 50, 52, 54, 55, 57, 58, 60, 61, 63, 65, 67, 68, 70, 71,73, 74, 76, 77, 79, 80, 82, 83, 85, 86, 88, 89, 91, 92, 94, 95, 97, 98,100, 101, 103, 104, 106, 107, 109, 110, 112 and 113.

According to another embodiment of the present invention, the presentinvention provides a composition for preventing or treating a diseaseassociated with HPV infection, the composition including, as an activeingredient, a nucleotide pool selected from the group consisting of: apool having nucleotide sequences of SEQ ID Nos: 2, 4, 8, 9, 12, and 15;a pool having nucleotide sequences of SEQ ID Nos: 18, 21, 29, and 32; apool having nucleotide sequences of SEQ ID Nos: 42, 45, 52, and 55; apool having nucleotide sequences of SEQ ID Nos: 58, 59, 63 and 65; apool having nucleotide sequences of SEQ ID Nos: 68, 71, 91 and 94; and apool having nucleotide sequences of SEQ ID Nos: 98, 100, 109 and 112.

According to the present invention, the present inventors have foundthat when the nucleotide sequence of the present invention is used as apool having particular combination, efficiency of inhibiting target geneexpression is considerably increased, when compared with the case wherea single sequence of RNA is used, so that a more outstanding therapeuticactivity to a disease associated with HPV infection is achieved.

According to a preferred embodiment of the present invention, a diseaseassociated with HPV infection to be treated by the composition of thepresent invention is selected from the group consisting of genitalwarts, vagina inflammation, pelvic inflammation and a cancer, and morepreferably, a cancer treated by the composition of the present inventionis selected from the group consisting of cervical cancer, vagina cancer,vulva cancer, anal cancer, penis cancer, tonsil cancer, pharynx cancer,larynx cancer, head and neck cancer and lung adenocarcinoma. Mostpreferably, the cancer to be treated by the composition of the presentinvention is cervical cancer.

The composition of the present invention may be prepared as apharmaceutical composition including a pharmaceutically effective amountof the nucleic acid molecule of the present invention.

As used herein, the term “pharmaceutically effective amount” means anamount sufficient to achieve the activity or efficacy of treating,alleviating, or preventing arthritis, as described above, of the presentinvention.

A pharmaceutically acceptable carrier, which is included in thepharmaceutical composition of the present invention, is one typicallyused in preparation, and includes, but not limited to, lactose,dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calciumphosphate, alginate, gelatin, calcium silicate, microcrystallinecellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc,magnesium stearate, cyclodextrin and a copolymer thereof, mineral oil,and so forth. The pharmaceutical composition of the present inventionmay further include a lubricant, a humectant, a sweetening agent, afavoring agent, an emulsifier, a suspending agent, and a preservingagent besides the components above. A suitable pharmaceuticallyacceptable carrier and a preparation are described in Remington'sPharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be orally orparenterally administered, and preferably parenterally administered. Forparenteral administration, intravenous infusion, subcutaneous infusion,intramuscular infusion, peritoneal infusion, topical administration,transdermal administration, and intraarticular administration may beused.

Suitable administration dose of the pharmaceutical composition of thepresent invention may be differentially prescribed depending on variousfactors such as a method for formulation, a mode of administration, age,weight, sex, and disease states, dietary of patients, time ofadministration, a route of administration, a secretion rate and reactionsusceptibility. A preferable administration dose of the pharmaceuticalcomposition of the present invention is 0.0001 to 100 mg/kg per day.

When the pharmaceutical composition of the present invention is used asan anti-cancer agent, the composition may be used as combination withthe anti-cancer composition typically used in the art. Morespecifically, the composition may be combinatorially administered withanti-cancer agents such as cisplatin or paclitaxel.

The pharmaceutical composition of the present invention is prepared in aunit dosage form by being formulated using a pharmaceutically acceptablecarrier and/or excipient, or prepared by being incorporated into amulti-dose container, according to a method by which a person withordinary skill in the technical field to which the present inventionbelongs could easily carry out. In this case, the formulation may be aform of a solution, suspension, or emulsion in an oil or an aqueousmedium, or extract, powder, granule, tablet, or capsule form, and mayfurther include a dispersing agent or a stabilizer.

According to more preferred embodiment of the present invention, thenucleic acid molecule of the present invention is included in a genedelivery system.

As used herein, the term “gene delivery system” means a mediator tointroduce a desired target gene in subject cells to express. The idealgene delivery system should be nontoxic to the human body, easily massproduced, and deliver efficiently the gene.

As used herein, the term “gene delivery” means delivering the gene intocells, and has the same meaning as cellular transduction of the gene. Atthe tissue level, the term gene delivery has the same meaning as spreadof the gene. Thus, the gene delivery system of the present invention maybe described as the gene transduction system and the gene spread system.

To prepare the gene delivery system of the present invention, thenucleotide sequence of the present invention is preferably presentwithin a suitable expression construct. In the expression construct, itis preferable that the nucleotide sequence of the present invention isoperatively linked to a promoter. As used herein, the term “operativelylinked to” means a functional binding between a regulatory sequence ofnucleic acid expression (for example, a promoter, a signal sequence, oran array at a transcription regulatory factor binding site) with othernucleic acid sequences, and the regulatory sequence thus regulatestranscription and/or translation of the other nucleic acid sequences. Inthe present invention, a promoter, which binds to the nucleotidesequence of the present invention, may be operated preferably in animalcells, and more preferably in mammalian cells to regulate transcriptionof relaxin gene, and includes, but not limited to a promoter derivedfrom mammalian virus and a promoter derived from a genome of mammaliancells such as mammalian cytomegalovirus (CMV) promoter, adenovirus latepromoter, vaccinia virus 7.5K promoter, SV40 promoter, tk promoter ofHSV, RSV promoter, EF1 alpha promoter, metallothionein promoter,beta-actin promoter, a promoter of human IL-2 gene, a promoter of humanIFN gene, a promoter of human IL-4 gene, a promoter of human lymphotoxingene, a promoter of human GM-CSF gene, and U6 promoter.

The gene delivery system of the present invention may be constructed invarious forms which are (i) a naked recombinant DNA molecule, (ii) aplasmid, (iii) a virus vector, and (iv) a liposome or a noisome formincluding the naked recombinant DNA molecule or the plasmid.

The nucleotide sequence of the present invention may be applied to wholegene delivery system used for typical gene therapy. Preferably, thenucleotide sequence of the present invention may be applied to aplasmid, adenovirus (Lockett L J, et al., Clin. Cancer Res.3:2075-2080(1997)), adeno associated virus (AAV, Lashford L S., et al.,Gene Therapy Technologies, Applications and Regulations Ed. A. Meager,1999), retrovirus (Gunzburg W H, et al., Retroviral vectors. GeneTherapy Technologies, Applications and Regulations Ed. A. Meager, 1999),lentivirus (Wang G. et al., J. Clin. Invest. 104(11):R55-62(1999)),herpes simplex virus (Chamber R., et al., Proc. Natl. Act. Sci USA92:1411-1415(1995)), vaccinia virus (Puhlmann M. et al., Human GeneTherapy 10:649-657(1999)), a liposome (Metho s in Molecular Biology, Vol199, S. C. Basu and M. Basu (Eds.), Human Press 2002) or a niosome.

Most preferably, the nucleotide sequence of the present invention isdelivered by using a cationic liposome.

According to another aspect of the present invention, the presentinvention provides a method for treating or preventing a diseaseassociated with HPV infection, the method including administering, to asubject, a pharmaceutical composition including: (a) a pharmaceuticallyeffective amount of one or more nucleotide sequences selected from thegroup consisting of sequences of SEQ ID Nos: 16, 22, 28, 34, 40, 66, 72,84, 90, and 108 and antisense nucleotide sequences thereof; and (b) apharmaceutically acceptable carrier.

According to another aspect of the present invention, the presentinvention provides a method for treating or preventing a diseaseassociated with HPV infection, the method including administering, to asubject, a pharmaceutical composition including: (a) a pharmaceuticallyeffective amount of one or more nucleotide sequences selected from thegroup consisting of sequences of SEQ ID Nos: 1, 7, 12, 16, 22, 28, 34,40, 46, 51, 56, 62, 66, 72, 78, 84, 90, 96, 102 and 108, and antisensenucleotide sequences thereof which have a modified backbone or one ormore modified bases; and (b) a pharmaceutically acceptable carrier.

According to another aspect of the present invention, the presentinvention provides a method for preventing or treating a diseaseassociated with HPV infection, the method including administering, to asubject, a pharmaceutical composition including: (a) a pharmaceuticallyeffective amount of nucleotide pool selected from the group consistingof: a pool having nucleotide sequences of SEQ ID Nos: 2, 4, 8, 9, 12,and 15; a pool having nucleotide sequences of SEQ ID Nos: 18, 21, 29,and 32; a pool having nucleotide sequences of SEQ ID Nos: 42, 45, 52,and 55; a pool having nucleotide sequences of SEQ ID Nos: 58, 59, 63 and65; a pool having nucleotide sequences of SEQ ID Nos: 68, 71, 91 and 94;and a pool having nucleotide sequences of SEQ ID Nos: 98, 100, 109 and112; and (b) a pharmaceutically acceptable carrier.

Since the method of the present invention uses the present compositiondescribed above, the features common to both the composition and themethod are not described herein to avoid excessive complexity in thespecification.

Advantageous Effects

The features and benefits of the present invention are summarized asfollows:

(a) The present invention is to provide a composition for preventing ortreating a disease associated with human papilloma virus (HPV)infection, more particularly, an HPV infection-associated cancer, andfurther more particularly a cervical cancer;

(b) The nucleotide sequence of the present invention, a sequence havinga modification in a base of the nucleotide sequence, and particularcombination thereof significantly inhibits expression of E6/E7 genes ofHPV type 16 or HPV type 18 viruses, and is thus usefully employed as acomposition or a method for efficiently treating a disease associatedwith HPV infection.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show images of results which verify improvement in stability(FIG. 1A), an increase in an effect at the molecular level of a protein(FIG. 1B), and an increase in an effect at the molecular level of mRNA(FIG. 1C) according to a substitutional modification of a residue of abase sequence in siRNA of HPV 16, and 18 types.

FIGS. 2A-2B show images of results which verify an excellent effect ofinducing cell senescence (FIG. 2A) and an excellent effect of killingcells (FIG. 2B) of siRNA having substituted residue in a base sequenceof HPV 18 type.

FIGS. 3A-3B are images showing the effect of inducing cell senescence bycombined treatment of the anti-cancer agent, cisplatin, and 426 siRNAhaving a substitution in a base sequence in a HeLa cervical cancer cellline infected with the HPV 18 type virus (FIG. 3A) and a microscopicallyobserved result thereof (FIG. 3B).

FIGS. 4A-4C show images of results which verify a therapeutic effect ofa pool of excellent siRNA having a substitution in a base sequence ofHPV 16, and 18 types, and a synergetic effect by combined therapy withcisplatin. FIGS. 4A, 4B, and 4C respectively show a cell-proliferationinhibiting effect, a cell-killing effect, and an effect at the molecularlevel of a protein.

FIGS. 5A-5B show images of results which verify an off-target effect ofsiRNA of HPV 18 type in cells and animals. FIGS. 5A and 5B respectivelyshow an effect of reducing IL-6 on cells and an effect of reducingINF-gamma in animals.

FIG. 6 shows an image of results which quantify 426 siRNA of HPV 18 typethrough stem-loop real-time PCT.

FIG. 7 shows an image of results which verify the fact that siRNAs invarious types of liposomes show the same cell-killing effect on cells.

FIGS. 8A-8C are images showing a synergistic effect by combinedtreatment of an anti-cancer agent and a pool of siRNAs, which have asubstitution in a base sequence and show the excellent effect, in ananimal experiment. FIGS. 8A, 8B, and 8C respectively show a variation ina size of a tumor of a mouse, an image of the tumor of the mouse, and avariation in body weight of the mouse.

MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in more detail withreference to examples. These examples are provided to only specificallydescribe the present invention, and it will be obvious to a personskilled in the art that the scope of the present invention is notlimited to the examples according to the essential features of thepresent invention.

EXAMPLE Experiment Method

Construction of siRNA of HPV 16, and 18 Types

siRNAs in Tables 1 and 2 below are obtained from Bioneer corporation(Korea) through customized production. In the table below, theunderlined siRNA sequence indicates a nucleotide substituted with a2′-O-Me modified nucleotide in which a methyl group is bound to aresidue of the base, and the thick italicized sequence indicates a basesubstituted with 2′-F modified nucleotide in which a hydroxyl group of aresidue of the base is substituted with fluoro.

TABLE 1 siRNA to HPV 16 type SEQ. ID. No. Sequence Note sequence5′-GCA AAG ACA UCU GGA CAA A-3′ HPV 16 Type 1 siRNA 366 sequence5′-GCA AAG ACA UCU GGA CAA A-3′ 2 sequence5′-GCA AAG ACA UCU GGA CAA A-3′ 3 sequence5′-UUU GUC CAG AUG UCU UUG C-3′ 4 sequence5′-UUU GUC CAG AUG UCU UUG C-3′ 5 sequence5′-UUU GUC CAG AUG UCU UUG C-3′ 6 sequence5′-UCA AGA ACA CGU AGA GAA A-3′ HPV 16 Type 7 siRNA 448 sequence5′-UCA AGA ACA CGU AGA GAA A-3′ 8 sequence5′-UUU CUC UAC GUG UUC UUG A-3′ 9 sequence5′-UUU CUC UAC GUG UUC UUG A-3′ 10 sequence5′-UUU CUC UAC GUG UUC UUG A-3′ 11 sequence5′-GAC CGG UCG AUG UAU GUC UUG-3′ HPV 16 Type 12 siRNA 497 sequence5′-GAC CGG UCG AUG UAU GUC UUG-3′ 13 sequence5′-AGA CAU ACA UCG ACC GGU CCA-3′ 14 sequence5′-AGA CAU ACA UCG ACC GGU CCA-3′ 15 sequence5′-GGA GCG ACC CAG AAA GTT A-3′ HPV 16 Type 16 siRNA 39 sequence5′-GGA GCG ACC CAG AAA GTT A-3′ 17 sequence 5′-GGA G

G A

 

AG AAA GTT A-3′ 18 sequence 5′-UAA CUU UCU GGG UCG CUC C-3′ 19 sequence5′-UAA CUU UCU GGG UCG CUC C-3′ 20 sequence5′-UAA CUU UCU GGG UCG CUC C-3′ 21 sequence5′-CAG AAA GTT ACC ACA GTT A-3′ HPV 16 Type 22 siRNA 48 sequence5′-CAG AAA GTT ACC ACA GTT A-3′ 23 sequence 5′-

AG AAA GTT A

 A C A GTT A-3′ 24 sequence 5′-UAA CUG UGG UAA CUU UCU G-3′ 25 sequence5′-UAA CUG UGG UAA CUU UCU G-3′ 26 sequence5′-UAA CUG UGG UAA CUU UCU G-3′ 27 sequence5′-GCA CAG AGC TGC AAA CAA C-3′ HPV 16 Type 28 siRNA 68 sequence5′-GCA CAG AGC TGC AAA CAA C-3′ 29 sequence 5′-G

A CAG AG

 TG

 AAA 

AA C-3′ 30 sequence 5′-GUU GUU UGC AGC UCU GUG C-3′ 31 sequence5′-GUU GUU UGC AGC UCU GUG C-3′ 32 sequence5′-GUU GUU UGC AGC UCU GUG C-3′ 33 sequence5′-GCA AAC AAC TAT ACA TGA T-3′ HPV 16 Type 34 siRNA 78 sequence5′-GCA AAC AAC TAT ACA TGA T-3′ 35 sequence 5′-G

A AA

 AA

 TAT A

A TGA T-3′ 36 sequence 5′-AUC AUG UAU AGU UGU UUG C-3′ 37 sequence5′-AUC AUG UAU AGU UGU UUG C-3′ 38 sequence5′-AUC AUG UAU AGU UGU UUG C-3′ 39 sequence5′-AGC AAA GAC ATC TGG ACA A-3′ HPV 16 Type 40 siRNA 365 sequence5′-AGC AAA GAC ATC TGG ACA A-3′ 41 sequence 5′-AG

 AAA GA

 AT

 TGG A

A A-3′ 42 sequence 5′-UUG UCC AGA UGU CUU UGC U-3′ 43 sequence5′-UUG UCC AGA UGU CUU UGC U-3′ 44 sequence5′-UUG UCC AGA UGU CUU UGC U-3′ 45 sequence5′-CAC CUA CAU UGC AUG AAU AUA-3′ HPV 16 Type 46 siRNA 573 sequence5′-CAC CUA CAU UGC AUG AAU AUA-3′ 47 sequence5′-UAU AUU CAU GCA AUG UAG GUG-3′ 48 sequence5′-UAU AUU CAU GCA AUG UAG GUG-3′ 49 sequence5′-UAU AUU CAU GCA AUG UAG GUG-3′ 50 sequence5′-CUU CGG UUG UGC GUA CAA AGC-3′ HPV 16 Type 51 siRNA 792 sequence5′-CUU CGG UUG UGC GUA CAA AGC-3′ 52 sequence5′-GCU UUG UAC GCA CAA CCG AAG-3′ 53 sequence5′-GCU UUG UAC GCA CAA CCG AAG-3′ 54 sequence5′-GCU UUG UAC GCA CAA CCG AAG-3′ 55

TABLE 2  siRNA to HPV 18 type SEQ. ID. No. Sequence Note sequence5′-CAA CCG AGC ACG ACA GGA A-3′ HPV 18 Type 56 siRNA 426 sequence 5′-

AA 

G AG

 A

G A

A GGA A-3′ 57 sequence 5′-CAA CCG AGC ACG ACA GGA A-3′ 58 sequence5′-UUC CUG UCG UGC UCG GUU G-3′ 59 sequence5′-UUC CUG UCG UGC UCG GUU G-3′ 60 sequence5′-UUC CUG UCG UGC UCG GUU G-3′ 61 sequence5′-CCA ACG ACG CAG AGA AAC A-3′ HPV 18 Type 62 siRNA 450 sequence5′-CCA ACG ACG CAG AGA AAC A-3′ 63 sequence5′-UGU UUC UCU GCG UCG UUG G-3′ 64 sequence5′-UGU UUC UCU GCG UCG UUG G-3′ 65 sequence5′-ACT GCA AGA CAT AGA AAT A-3′ HPV 18 Type 66 siRNA 72 sequence5′-ACT GCA AGA CAT AGA AAT A-3′ 67 sequence 5′-A

T G

A AGA 

T AGA AAT A-3′ 68 sequence 5′-UAU UUC UAU GUC UUG CAG U-3′ 69 sequence5′-UAU UUC UAU GUC UUG CAG U-3′ 70 sequence5′-UAU UUC UAU GUC UUG CAG U-3′ 71 sequence5′-GTA TAT TGC AAG ACA GTA T-3′ HPV 18 Type 72 siRNA 97 sequence5′-GTA TAT TGC AAG ACA GTA T-3′ 73 sequence 5′-GTA TAT TG

 AAG A

A GTA T-3′ 74 sequence 5′-AUA CUG UCU UGC AAU AUA C-3′ 75 sequence5′-AUA CUG UCU UGC AAU AUA C-3′ 76 sequence5′-AUA CUG UCU UGC AAU AUA C-3′ 77 sequence5′-GCA AGA CAG TAT TGG AAC T-3′ HPV 18 Type 78 siRNA 103 sequence5′-GCA AGA CAG TAT TGG AAC T-3′ 79 sequence 5′-G

A AGA 

AG TAT TGG AA

 T-3′ 80 sequence 5′-AGU UCC AAU ACU GUC UUG C-3′ 81 sequence5′-AGU UCC AAU ACU GUC UUG C-3′ 82 sequence5′-AGU UCC AAU ACU GUC UUG C-3′ 83 sequence5′-ATT GGA ACT TAC AGA GGT A-3′ HPV 18 Type 84 siRNA 113 sequence5′-ATT GGA ACT TAC AGA GGT A-3′ 85 sequence5′-ATT GGA ACT TAC AGA GGT A-3′ 86 sequence5′-UAC CUC UGU AAG UUC CAA U-3′ 87 sequence5′-UAC CUC UGU AAG UUC CAA U-3′ 88 sequence5′-UAC CUC UGU AAG UUC CAA U-3′ 89 sequence5′-CTC CAA CGA CGC AGA GAA A-3′ HPV 18 Type 90 siRNA 448 sequence5′-CTC CAA CGA CGC AGA GAA A-3′ 91 sequence 5′-CT

 CAA 

GA 

G

 AGA GAA A-3′ 92 sequence 5′-UUU CUC UGC GUC GUU GGA G-3′ 93 sequence5′-UUU CUC UGC GUC GUU GGA G-3′ 94 sequence5′-UUU CUC UGC GUC GUU GGA G-3′ 95 sequence5′-ACG CAG AGA AAC ACA AGT A-3′ HPV 18 Type 96 siRNA 456 sequence5′-ACG CAG AGA AAC ACA AGT A-3′ 97 sequence5′-ACG CAG AGA AAC ACA AGT A-3′ 98 sequence5′-UAC UUG UGU UUC UCU GCG U-3′ 99 sequence5′-UAC UUG UGU UUC UCU GCG U-3′ 100 sequence5′-UAC UUG UGU UUC UCU GCG U-3′ 101 sequence5′-GCA GAG AAA CAC AAG TAT A-3′ HPV 18 Type 102 siRNA 458 sequence5′-GCA GAG AAA CAC AAG TAT A-3′ 103 sequence 5′-G

A GAG AAA 

A

 AAG TAT A-3′ 104 sequence 5′-UAU ACU UGU GUU UCU CUG C-3′ 105 sequence5′-UAU ACU UGU GUU UCU CUG C-3′ 106 sequence5′-UAU ACU UGU GUU UCU CUG C-3′ 107 sequence5′-CAG AGA AAC ACA AGT ATA A-3′ HPV 18 Type 108 siRNA 459 sequence5′-CAG AGA AAC ACA AGT ATA A-3′ 109 sequence 5′-

AG AGA AA

 A

A AGT ATA A-3′ 110 sequence 5′-UUA UAC UUG UGU UUC UCU G-3′ 111 sequence5′-UUA UAC UUG UGU UUC UCU G-3′ 112 sequence5′-UUA UAC UUG UGU UUC UCU G-3′ 113

Evaluation of Stability of siRNA

siRNA was mixed with 10% fetal bovine serum or 10% human serum whilestaying at 37□, and samples were taken in a time based manner. Then,samples were quick frozen and stored at −70□. Collected samples weresubjected to electrophoresis for one and a half hours on 12%polyacrylamide gel at 50 V followed by Et-Br staining for UVmeasurement.

Cell Culture and Transduction of siRNA

A cervical cancer cell, a HeLa cervical cancer cell line (ATCC CCL-2)infected with HPV 18 type virus, or SiHa (ATCC HTB-35) or CaSki (ATCCCRL-1550) cervical cancer cell line infected with HPV 16 type virus wasseeded on a 6-well plate in the cell number of 2×10⁵ or 1.6×10⁵, andrespectively cultured in RPMI1640 or DMEM medium having 10% fetal bovineserum added thereto for 24 hours under the condition of 37□, and 5% CO₂.After culturing for 24 hours in the medium to attach cells to a surfaceof the culture plate, unmodified siRNA as a control and an siRNAoligonucleotide modified by the method described above as anexperimental group (20 nM for each), were transduced by using DharmaFECT1 (Dharmacon, USA), and the resultant was cultured for 24 hours.

Anti-Cancer Agent Treatment

A HeLa or Caski cell line, which was seeded in a 6-well plate in 2×10⁵cells or 1.5×10⁵ cells and then cultured for a day by the method asdescribed above, was transduced with siRNA. Then, each transduced cellline was treated with cisplatin (CDDP) in a final concentration of 2.5uM, and cultured.

Cell Senescence-Associated β-Galactosidase (SA-β-Gal) ActivityMeasurement

By the method as described above, a HeLa or Caski cell line wastransduced with siRNA, alone or in combination with an anti-canceragent, and then cultured for a day. By using the cell senescence assaykit (BioVision, USA), cells were washed with PBS and treated withSA-β-gal staining solution for 12 hours at 37□. Cells stained in bluewere observed by using a general optical microscope with themagnification of 100 to 200 times.

Measurement of Cell Death by Using Flow Cytometry

By the method as described above, a HeLA or Caski cell line wastransduced with siRNA, alone or in combination with an anti-canceragent, and then cultured for a day. The cells were stained with andreacted to Annexin V and propidium iodide (PI) reagents for 30 minutesat room temperature by using cell death assay kit (BD, USA), andthereafter cell death was evaluated by using a flow cytometry.

Investigation of Influence of siRNA Treatment

By the method as described above, a HeLA or Caski cell line wastransduced with siRNA, alone or in combination with an anti-canceragent, and then cultured for a day. To observe a change in a protein,cells were disrupted by adding RIPA cell lysis buffer [150 mM NaCl, 10mM Tris-HCl (pH 7.4), 5 mM EDTA, 0.1% SDS, 0.5% deoxycholate and 1%NP-40]. Then, variation in protein level was observed through thegeneral western-blot method. Anti-TP53, anti-E7, and anti-actin mouseantibody were purchased from Santa Cruz (USA), diluted to 1:1000, andused. The goat anti-mouse IgG HRP conjugated antibody was purchased fromJackson Laboratories (USA), diluted to 1:3000, and used.

Further, to observe variation in mRNA level, cells were disrupted byusing a Trizol solution (Invitrogen, USA), and RNA was detected passingthrough ethanol purification to thereby observe variation in mRNA levelthrough the general real-time polymerase chain reaction (PCR) method.

Investigation of Influence of Off-Target Effect of siRNA

A HeLA or Caski cell line was seeded on a 6-well plate, and respectivelycultured for 24 hours in RPMI1640 or DMEM medium having 10% fetal bovineserum under the condition of 37□, and 5% CO₂. β-gal siRNA, which was apositive control, and siRNA were transduced and cultured, and then themedium was collected to perform the general IL-6 (BD, USA) ELISA method.

A mouse at the age of 6 weeks was intravenously injected with β-galsiRNA as a positive control, siRNA as a negative control, and siRNA, andthe reaction was proceeded for 6 hours. Thereafter, blood was collectedfrom the mouse, and serum was separated to perform INF-gamma (BD, USA)ELISA.

Stem-Loop Real-Time PCR to Quantify siRNA

A rat weighing about 260 to 300 g (at the age of 4 weeks) wasintravenously injected with siRNA, and then blood of the rat wascollected to separate plasma. The separated plasma was diluted in 0.25%triton X-100 buffer. cDNA was synthesized by using Taqman microRNAReverse Transcription kit (Applied Biosystem, USA), and quantified bythe real-time PCR method to detect siRNA.

Animal Test of siRNA

A female nude mouse was xenografted with 5×10⁶ of HeLa cells of HPV 18type, and generation of cancer cells was evaluated 10 days later. Then,3 mg/kg of siRNA to be used was intravenously injected to tails at the2-3 day interval. Cisplatin (2 mg/kg) and paclitaxel (4 mg/kg) wererepeatedly injected 9 times by an intraperitoneal injection at 3-4 dayinterval. The size of a tumor was measured at 2-3 day interval.

Experimental Result

Variation in siRNA Treatment Concentration and Treatment Number

For a Caski or HeLa cell line infected with HPV 16 or 18 type, whenperforming siRNA transduction and an anti-cancer agent treatment, aloneor in combination, in conventional technique, efficacy of siRNA wasshowed in successive treatment for long period at a high concentration(100 nM), while the modified siRNA of the present invention exhibitedexcellent efficacy in single treatment for short period at a lowconcentration (20 nM).

Stability Test of siRNA Having Substituted Residue

siRNAs of combination 51 to 54, obtained by the method described above,were mixed with 10% human serum. Then, each siRNA was taken in atime-based manner at the temperature of 37□, and stored at −70□.Thereafter, gel electrophoresis was performed on 12% polyacrylamide gel.The resultant was stained with Et-Br, and measured with UV.Consequently, as shown in FIG. 1a , siRNA of combination 51 having anunsubstituted residue in a base sequence was disappeared within twohours, while siRNAs of combination 52 to 54 having substituted residuein a base sequence were remained at least 4 hours, indicating aconsiderable increase in stability. In particular, combination showedthe most outstanding stability which lasts over 24 hours.

Effect of siRNA Having Substituted Residue at the Molecular Level

By the method described above, a HeLa cell line was transduced withsiRNAs of combination 44 to 50, mock and GFP siRNA, and then variationin TP 53, and E7 protein levels was evaluated, while using actin as ahousekeeping gene, wherein siRNAs of combination 44 to 50 were siRNAs ofHPV 18 type, and mock and GFP were controls. As shown in FIG. 1b ,variation in TP53 and E7 protein level of combination 44 was comparedwith that of combination 45 to 50, wherein combination 44 has anunsubstituted residue in a base sequence, and combination 45 to 50 has asubstituted residue in a base sequence. It was proven that efficacy ofcombination 48 to increase TP53 protein expression and to reduce E7protein expression was superior to other sequences.

Further, for siRNA 497 of HPV 16 type, a Caski cell line was transducedwith combination 12 to 15 by the method described above, and then mRNAwas extracted to evaluate mRNA expression levels of E6 and P21.Consequently, as shown in FIG. 1c , it has been proven that combination13 reduced by 60% or more of E6 mRNA and increased by 1100% or more ofp21 mRNA with respect to the control, wherein, combination 13 has asubstituted residue in a base sequence. It has been also evaluated thatcombination 13 was superior to other sequences comparing withcombination 12 which has an unsubstituted residue in a base sequence.

Cell Senescence-Inducing Effect of siRNA Having Substituted Residue

SA β-Gal activity of a HeLA or Caski cell line, which was treated by themethod as described above, was measured. Consequently, in FIG. 2a , inthe cases where HPV 18E6/E7 siRNA and combination 51 were transduced,the SA-β Gal activity was incased by about 10 to 20 times, while theSA-β Gal activity for combination 54 was increased by 50 times or morecomparing with that of the control siRNA, wherein HPV 18E6/E7 siRNA wasused in the previous invention; combination 51 consisted of 450 siRNAhaving an unsubstituted residue in a base sequence; and combination 54consisted of siRNA having a substituted residue in a base sequence. Theresult showed that the cell senescence effect of combination 54 havingsubstation in a base sequence was considerably better than that ofcombination 51 having no substitution of a base sequence.

Cell-Killing Effect of siRNA Having Substituted Residue

By the method described above, a cell-killing effect was measured byusing a flow cytometry. As a result obtained by evaluating thecell-killing effect by comparing HPV 18E6/E7, 18E6 siRNA, combination48, and combination 54 (which have a substituted residue in a basesequence) with the control, it has been proven that HPV 18E6/E7 and 18E6siRNA groups showed cell-killing effect of only 15 to 20%, while siRNAgroups having a substituted residue showed about 60% or more ofcell-killing effect, with respect to the control (FIG. 2b ). The resultshowed that siRNA sequences having substitution in a base sequenceshowed more significant cancer cell-killing effect than typical siRNA.

Effect of Combined Treatment of Cisplatin and siRNA Having SubstitutedResidue

As a result of measuring SA β-Gal activity by treating a HeLa cell linewith cisplatin and combination 48 having a substituted residue in a basesequence by the method as described above, as shown in FIGS. 3a and 3b ,cells were stained in blue in cell lines respectively treated withcisplatin and combination 48 alone, showing SA β-Gal activity. However,almost of cells were stained in dark blue which indicates strong SAβ-Gal activity for the combined treatment group of combination 48 andcisplatin. The result showed that the combined treatment group exhibitedmuch superior cell senescence effect to the mono-treatment group ofsiRNA or cisplatin.

Effect of Inhibiting Cell Proliferation by Mono-Treatment or CombinedTreatment of siRNA Pool

By the method described above, HPV 16 type Caski cell lines wererespectively treated with 20 mM of siRNA combination 2, 9, and 13 alone,wherein the siRNA combination 2, 9, and 13 has substitution in a basesequence of HPV 16 type, and HPV 16 type Caski cell lines weretransduced with: the pool of combination 2 and combination 9 (10 nM, foreach); the pool combination 2 and combination 13 (10 nM, for each); thepool of combination 9 and combination 13 (10 nM, for each); and pool ofcombination 2, combination 9, and combination 13 (7 nM, for each). Then,cell number was measured 24 hours later. Consequently, as shown FIG. 4a, cell proliferation was reduced in the mono treatment groups of eachsiRNA having substitution in a base sequence, and the siRNA pooltreatment group in a similar manner. Particularly, although each siRNAwas treated in an amount of 7 nM, the pool of 3 types of siRNA showedthe effect equivalent to the case where 20 nM of each siRNA was treated.

Combination of the siRNA pool used herein was shown in Table 3 below,and siRNA pool shown in Table 4 below includes a mixture of two or moreof combination having particularly higher cell proliferation inhibitingeffects.

TABLE 3 Combination of siRNA Number of combination Sense Antisense notecombination1 sequence 1 sequence 4 HPV 16 type combination2 sequence 2sequence 4 siRNA 366 combination3 sequence 2 sequence 5 combination4sequence 2 sequence 6 combination5 sequence 3 sequence 4 combination6sequence 3 sequence 5 combination7 sequence 3 sequence 6 combination8sequence 7 sequence 9 HPV 16 type combination9 sequence 8 sequence 9siRNA 448 combination10 sequence 8 sequence 10 combination11 sequence 8sequence 11 combination12 sequence 12 sequence 14 HPV 16 typecombination13 sequence 12 sequence 15 siRNA 497 combination14 sequence13 sequence 14 combination15 sequence 13 sequence 15 combination16sequence 16 sequence 19 HPV 16 Type combination17 sequence 17 sequence20 siRNA 39 combination18 sequence 18 sequence 21 combination19 sequence18 sequence 21 combination20 sequence 22 sequence 25 HPV 16 Typecombination21 sequence 23 sequence 25 siRNA 48 combination22 sequence 24sequence 26 combination23 sequence 24 sequence 27 combination24 sequence28 sequence 31 HPV 16 Type combination25 sequence 29 sequence 32 siRNA68 combination26 sequence 30 sequence 32 combination27 sequence 30sequence 33 combination28 sequence 34 sequence 34 HPV 16 Typecombination29 sequence 35 sequence 37 siRNA 78 combination30 sequence 36sequence 38 combination31 sequence 36 sequence 39 combination32 sequence40 sequence 43 HPV 16 Type combination33 sequence 41 sequence 44 siRNA365 combination34 sequence 42 sequence 44 combination35 sequence 42sequence 45 combination36 sequence 46 sequence 48 HPV 16 Typecombination37 sequence 46 sequence 49 siRNA 573 combination38 sequence47 sequence 48 combination39 sequence 47 sequence 50 combination40sequence 51 sequence 53 HPV 16 Type combination41 sequence 51 sequence55 siRNA 792 combination42 sequence 52 sequence 54 combination43sequence 52 sequence 55 combination44 sequence 56 sequence 59 HPV 18type combination45 sequence 57 sequence 59 siRNA 426 combination46sequence 57 sequence 60 combination47 sequence 57 sequence 61combination48 sequence 58 sequence 59 combination49 sequence 58 sequence60 combination50 sequence 58 sequence 61 combination51 sequence 62sequence 64 HPV 18 type combination52 sequence 62 sequence 65 siRNA 450combination53 sequence 63 sequence 64 combination54 sequence 63 sequence65 combination55 sequence 66 sequence 69 HPV 18 Type combination56sequence 67 sequence 70 siRNA 72 combination57 sequence 67 sequence 69combination58 sequence 68 sequence 71 combination59 sequence 72 sequence76 HPV 18 Type combination60 sequence 73 sequence 75 siRNA 97combination61 sequence 73 sequence 77 combination62 sequence 74 sequence77 combination63 sequence 78 sequence 83 HPV 18 Type combination64sequence 79 sequence 82 siRNA 103 combination65 sequence 80 sequence 81combination66 sequence 80 sequence 82 combination67 sequence 84 sequence89 HPV 18 Type combination68 sequence 85 sequence 87 siRNA 113combination69 sequence 86 sequence 88 combination70 sequence 86 sequence89 combination71 sequence 90 sequence 95 HPV 18 Type combination72sequence 91 sequence 94 siRNA 448 combination73 sequence 92 sequence 93combination74 sequence 92 sequence 95 combination75 sequence 96 sequence99 HPV 18 Type combination76 sequence 97 sequence 99 siRNA 456combination77 sequence 98 sequence 100 combination78 sequence 98sequence 101 combination79 sequence 102 sequence 107 HPV 18 Typecombination80 sequence 103 sequence 106 siRNA 458 combination81 sequence103 sequence 107 combination82 sequence 104 sequence 105 combination83sequence 108 sequence 113 HPV 18 Type combination84 sequence 109sequence 112 siRNA 459 combination85 sequence 110 sequence 112combination86 sequence 110 sequence 113

TABLE 4 siRNA Pool Number Number of com- of pool Sense Antisensebination Note SP1 sequence 2 sequence 4 2 HPV 16 type sequence 8sequence 9 9 siRNAs 366/ sequence 12 sequence 15 13 448/497 SP2 sequence18 sequence 21 18 HPV16 type sequence 29 sequence 32 25 siRNAs 39/68 SP3sequence 42 sequence 45 35 HPV16 type sequence 52 sequence 55 43 siRNAs365/792 SP4 sequence 58 sequence 59 48 HPV 18 type sequence 63 sequence65 54 siRNAs 426/450 SP5 sequence 68 sequence 71 58 HPV18 type sequence91 sequence 94 72 siRNAs 72/448 SP6 sequence 98 sequence 100 77 HPV18type sequence 109 sequence 112 84 siRNAs 456/459

Cell-Killing Effect of Mono-Treatment and Combined Treatment of siRNAPool

By the method described above, an HPV 18 type HeLa cell line was treatedwith combination 48 and combination 54 (20 nm, for each) wherein,combination 48 and combination 20 have siRNA of HPV 18 type havingsubstation in a base sequence, and transduced with the 10 nM of the SP4pool, which is the pool of siRNA of 5 nM of combination 48 and 5 nM ofcombination 54. After 24 hours, the cell line was stained with Annexin Vand propidium iodide, and cell-killing effect was measured by using aflow cytometry. Consequently, as shown in FIG. 4b , both themono-treatment groups of siRNA, which have substitution in a basesequence, and the siRNA pool (SP4) showed the effect of killing 80% ormore of cells. As a result, it has been proven that 20 nM of the siRNAmono-treatment group showed the cell-killing effect similar to that of10 nM of the siRNA pool, indicating that the siRNA pool was better.

Effect of siRNA Pool in Mono- and Combined Treatment at Molecular Level

By the method described above, in a HPV 16 type CaSki cell line, thewestern-bolt method was used to compare a TP53 protein expression levelfor a combined treatment group of cisplatin and combination 2, 9, 13; amono-treatment group of cisplatin; a combined treatment group ofcisplatin and combination 2 and 9; a combined treatment group ofcisplatin and combination 2 and 13; a combined treatment group ofcisplatin and combination 9 and 13; and a combined treatment group ofcisplatin and pool SP1 (a pool of combination 2, combination 9, andcombination 13), wherein combination 2, 9, and 13 have substitution in abase sequence. Consequently, as shown in FIG. 4C, among combinedtreatment groups of siRNA pools and cisplatin, the highest increase in aTP53 protein expression level was shown in the pool SP1 which wastreated with low concentration of 7 nM.

The result showed that since the pool selectively consisted of siRNAwhich was competent and efficient while mimicking features ofnaturally-occurring siRNA pool, it is possible to mix and use in aconcentration lower than the concentration of the typical treatment, andreduce off-target effect.

Off-Target Effect of siRNA

By the method described above, HPV 18 type HeLa cell line was transducedwith β-gal siRNA as a positive control, combination 44 having anunsubstituted residue, and combination 48 having a substituted residuein a base sequence. Then, an immune response experiment of IL-6 wasperformed. Consequently, as shown in FIG. 5a , immune response of IL-6was increased in the positive control and combination 44, while immuneresponse of IL-6 was reduced to ½ level of the positive control forcombination 48 having a substituted residue in a base sequence.

Further, a mouse at the age of 6 weeks was intravenously injected withβ-gal siRNA and HPV 18 type siRNA combination 44 and 48 which ariseimmune response, and reacted for 6 hours to perform an immune responseexperiment of INF-gamma (FIG. 5b ). Although immune response wasobserved in the positive control β-gal siRNA and combination 44 whichwas expressed by an increase in the INF-gamma level, immune response wasnot observed in combination 48 since INF-gamma in combination 48 showeda similar level to that of the negative control and did not increased.

Thus, it has been found that immune response was reduced in the siRNAtreatment group having a substituted residue comparing with that siRNAhaving an unsubstituted residue does.

Pharmacokinetic Experiment of siRNA Having Substituted Residue

By the method described above, a rat was intravenously injected with HPV18 type siRNA combination 44 and combination 48, and blood was collectedin a time-based manner. Then, plasma was separated to quantify siRNAthrough stem-loop real-time PCR method. Consequently, as shown in FIG.6a , half-life of combination 48 was at least twice longer than that ofcombination 44 meaning that combination 48, which has a substitutedresidue in a base sequence, is more stable in vivo.

Effect of siRNA in Various Types of Liposome

By the method described above, a HPV 18 type HeLa cell line wastransduced with siRNA by using commercially available Dharmafect(Dharmacon), Oligofectamine and Lipofectamine 2000 (Invitrgen) drugdelivery systems and a cationic liposome prepared by the presentinventors. After 24 hours, cell number was measured to evaluate theeffect of inhibiting cell proliferation. Consequently, as shown in FIG.7a , siRNA was effectively delivered to a cell line infected with HPV invarious drug delivery systems.

Effect of Combined Treatment of siRNA Pool and Anti-Cancer Agent

By the method described above, cancer cells were xenografted to a mouse.After 10 days, generation of cancer cells was evaluated. Then, siRNA andan anti-cancer agent were repeatedly injected 9 times, and the size of atumor was measured at 2-3 day interval. Consequently, as shown in FIG.8a , the combined treatment group of the anti-cancer drug and SP4 (apool of combination 48 and 54) showed the significantly outstandingtherapeutic effect than the combined treatment group of the anti-cancerdrug and the pool of combination 44 and 51. It has been found that SP4having a substituted residue in a base sequence showed better efficacyand effect than the siRNA pool of combination 44 and 51 having aunsubstituted residue in a base sequence. Moreover, as shown in FIG. 8b, when compared a size of tumors of a mouse of the anti-cancer agentadministration group of cisplatin and paclitaxel with that of thecombined treatment group of the anti-cancer agent and SP4 pool on day17, it has been proven that there are large differences in the size andstate. Also, as shown in FIG. 8c , a result obtained by observingamounts of variation in body weight of the mouse on day 9 and 28 showedno reduction in body weight caused by toxicity. Thus, it has beendetermined that there was no side effect caused by toxicity of siRNA.

Hitherto, specific features of the present invention are described indetail. However, it would be apparent to a person skilled in the artthat the specific description is preferable embodiment only, and thescope of the invention is not limited thereto. Therefore, substantialscope of the present invention would be defined by accompanying claimsand equivalents thereof.

1-26. (canceled)
 27. A method for preventing or treating a diseasecaused by HPV infection, the method comprising administering, to asubject, a pharmaceutical composition including: (a) a pharmaceuticallyeffective amount of nucleotide pool selected from the group consistingof: a pool having nucleotide sequences of SEQ ID Nos: 2, 4, 8, 9, 12,and 15; or a pool having nucleotide sequences of SEQ ID Nos: 58, 59, 63and 65; and (b) a pharmaceutically acceptable carrier.
 28. The method ofclaim 27, wherein, the disease caused by HPV infection is selected fromthe group consisting of genital warts, vagina inflammation, pelvicinflammation and a cancer.
 29. The method of claim 28, wherein, thecancer is selected from the group consisting of cervical cancer, vaginacancer, vulva cancer, anal cancer, penis cancer, tonsil cancer, pharynxcancer, larynx cancer, head and neck cancer and lung adenocarcinoma. 30.A method for treating or preventing a disease caused by HPV infection,the method comprising administering, to a subject, a pharmaceuticalcomposition including: (a) a pharmaceutically effective amount of one ormore nucleotide pair selected from the group consisting of: (i) anucleotide pair of sequences comprising SEQ ID NOs: 2 and 4,respectively, (ii) a nucleotide pair of sequences comprising SEQ ID NOs:8 and 9, respectively, (iii) a nucleotide pair of sequences comprisingSEQ ID NOs: 12 and 15, respectively, (iv) a nucleotide pair of sequencescomprising SEQ ID NOs: 13 and 14, respectively, (v) a nucleotide pair ofsequences comprising SEQ ID NOs: 13 and 15, respectively, (vi) anucleotide pair of sequences comprising SEQ ID NOs: 57 and 59,respectively, (vii) a nucleotide pair of sequences comprising SEQ IDNOs: 57 and 60, respectively, (viii) a nucleotide pair of sequencescomprising SEQ ID NOs: 58 and 59, respectively, (ix) a nucleotide pairof sequences comprising SEQ ID NOs: 58 and 60, respectively, (x) anucleotide pair of sequences comprising SEQ ID NOs: 58 and 61,respectively, and (xi) a nucleotide pair of sequences comprising SEQ IDNOs: 63 and 65; and (b) a pharmaceutically acceptable carrier.
 31. Themethod of claim 30, wherein, the nucleotide is siRNA.
 32. The method ofclaim 30, wherein, the disease caused by HPV infection is selected fromthe group consisting of genital warts, vagina inflammation, pelvicinflammation and a cancer.
 33. The method of claim 30, wherein, thecancer is selected from the group consisting of cervical cancer, vaginacancer, vulva cancer, anal cancer, penis cancer, tonsil cancer, pharynxcancer, larynx cancer, head and neck cancer, and lung adenocarcinoma.